四种牛病毒性呼吸道病一步法多重PCR方法的建立与应用  

Establishment and application of a one-step multiplex PCR method for four bovine viral respiratory diseases

作　　者：张闫 郭福强 刘小琛 段文龙 王利丽 白和平 陶勇 赵桂新 侯冠欣 张志强[1] 史秋梅[1] ZHANG Yan;GUO Fu-qiang;LIU Xiao-chen;DUAN Wen-long;WANG Li-li;BAI He-ping;TAO Yong;ZHAO Gui-xin;HOU Guan-xin;ZHANG Zhi-qiang;SHI Qiu-mei(Hebei Key Laboratory of Preventive Veterinary Medicine,Hebei Normal University of Science and Technology,Qinhuangdao 066004,China)

机构地区：[1]河北科技师范学院河北省预防兽医重点实验室,河北秦皇岛066004

出　　处：《中国兽医科学》2023年第11期1390-1397,共8页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31802186);河北省现代农业产业技术体系创新团队肉牛疫病防控岗位项目(HBCT2023-190201);河北省高等学校科学技术研究项目(BJ2021055);河北省重点研发计划项目(19226629D)。

摘　　要：牛病毒性呼吸道病(BRD)是一种世界性的并且由多个因素引起的传染病,也是危害我国牛养殖业的主要因素。本研究分别选取保守基因IBRV gB基因、BCoV N基因、BRSV F基因、BPIV3 HN基因,设计特异性引物,建立了IBRV、BCoV、BRSV、BPIV3一步法多重PCR检测方法,并对一步法多重PCR方法反应体系与反应程序进行优化。一步法多重PCR的退火温度、延伸时间、循环数、引物浓度、酶用量分别为60℃、30 s、30次、0.6μmol/L、1.6μL。该方法对IBRV、BRSV、BCoV、BPIV3的最低检测限分别为3.72×10^(-3)、3.14×10^(-6)、2.86×10^(-4)、2.92×10^(-6)ng/μL。利用所建立的一步法多重PCR方法对临床90份样品进行检测,与经典单重PCR检测方法相比,阳性符合率为100%。Bovine viral respiratory disease(BRD)is a worldwide and multifactorial infectious disease,and a major factor affecting the cattle breeding industry in China.In this study,the conserved genes IBRV gB gene,BCoV N gene,BRSV F gene and BPIV3 HN gene were selected and specific primers were designed to establish a one-step multiplex PCR assay for IBRV,BCoV,BRSV and BPIV3.The reaction system and procedure of one-step multiplex PCR method were optimized,and the annealing temperature,extension time,number of cycles,primer concentration and enzyme content were 60℃,30 s,30 times,0.6μmol/L and 1.6μL,respectively.The minimum detection limits of the method for IBRV,BRSV,BCoV and BPIV3 were 3.72×10^(-3),3.14×10^(-6),2.86×10^(-4),and 2.92×10^(-6)ng/μL,respectively.Using the established one-step multiplex PCR method to detect 90 clinical samples,compared with the classical single-plex PCR method,the positive coincidence rate was 100%.

关 键 词：一步法多重PCR 牛传染性鼻气管炎病毒 牛呼吸道合胞体病毒 牛冠状病毒 牛副流感病毒3型 

分 类 号：S852.653[农业科学—基础兽医学]